Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y,del156-157/R158G, and Del143-145) in settled solids using digital RT-PCR v2

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one each for HV69-70, E484K/N501Y, del156-157/R158G, and Del143-145) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA v2

Please note that while this protocol is for TNA extraction using the Perkin Elmer Chemagic 360, RNA extraction with resuspended solids from this protocol has been verified to perform well using the Kingfisher MagMax kit as another high throughput, automated option and two manual Qiagen kits - the All Prep Powerviral DNA/RNA Kit and the Qiamp Viral RNA Mini Kit. This process instruction describes the steps for purification of nucleic acids from wastewater solids and preparation for downstream quantitative analysis with Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR). Due to the large quantities of substances that have inhibitory effects on PCR in wastewater samples, a subsequent PCR inhibitor removal step is required after nucleic acid purification. Both steps of the process are carried out in a 96-well plate format. This method uses the resuspended solids generated using this protocol: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. RNA purification is carried out using a kit optimized for the purification of viral on for the Perkin Elmer Chemagic 360. Although only RNA is used in the downstream applications from this protocol, DNA is also eluted in this process. A crucial component of the purification kit are the magnetic particles coated with poly vinyl alcohol (M-PVA Magnetic Beads) which have a hydrophilic surface giving them an affinity for nucleic acids but not many other biological molecules. The workflow involves binding nucleic acids in a sample to the beads which are then transferred through a series of wash buffers to remove debris with a robotic head with magnetic rods. The OneStep PCR Inhibitor Removal Kits are PCR inhibitor clean up kits that contain all the components needed for efficient removal of contaminants that can inhibit downstream enzymatic reactions (e.g. PCR and RT) from DNA and RNA preparations. The column matrices in these PCR inhibitor clean up kits have been specifically designed for the efficient removal of polyphenolic compounds, humic/fulvic acids, tannins, melanin, etc. from the most impure DNA and RNA preparations. This process instruction applies to extraction of RNA from wastewater samples using the Chemagic™ Viral DNA/RNA 300 Kit H96 for the Perkin Elmer Chemagic 360 followed by PCR Inhibitor Removal with the Zymo OneStep-96 PCR Inhibitor Removal Kit.

Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y, and del156-157/R158G) in settled solids using digital RT-PCR v2

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one for HV69-70, one for E484K/N501Y, and one for del156-157/R158G) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR v2

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

Deteksi Brucella abortus dari Sampel Darah-Utuh dengan Uji Polymerase Chain Reaction Tanpa Ekstraksi DNA

Brucellosis is a zoonotic disease that cause a significant economic losses for cattle industries worldwide. A rapid, precise and accurate diagnosis technique for diagnosis of brucellosis in all stages of the infection is definitely required. Blood-samples are widely used for PCR-based DNA analysis because they are easily collected, handled, and processed. Direct PCR analysis without DNA extraction has been attempted to reduce time and costs for routine analysis. This approach is promising but is still limited by the presence of PCR inhibitors that is naturally found in the blood samples. The objective of this study was to compare the effectivity of direct PCR technique with or without DNA extraction for detection of Brucella abortus in the blood samples. Three whole-blood samples from brucella infected dairy cattle and five whole-blood samples from beef cattle that having abortion were used as samples in this study. A pair of bcsp31 primers and IS711 primers were used for amplification of genus-specific and species-specific of Brucella. The results showed that amplicon in the position of 223 bp and 498 bp that are specific for B. abortus were detected from all of the samples that were analyzed on 1.5% agarose gels. Based on the result it could be concluded that direct PCR analyses without DNA extraction is a sensitive, specific, simple, rapid and inexpensive assay for detecting B. abortus in the whole blood samples for either dairy or beef cattle and therefore it could improve the existing surveillance and control programs for brucellosis. Keywords : brucellosis; direct PCR; PCR inhibitor; whole-blood sample; without DNA extraction Abstrak Brucellosis adalah penyakit zoonosis yang menyebabkan kerugian ekonomi yang signifikan bagi industri ternak di seluruh dunia. Teknik diagnosis yang cepat, tepat dan akurat yang dapat digunakan untuk diagnosis brucellosis pada semua tahap infeksi sangat diperlukan. Sampel darah banyak digunakan untuk analisis PCR berbasis DNA karena mudah untuk dikoleksi, ditangani, dan diproses. Metoda PCR langsung tanpa didahului dengan ekstraksi DNA dikembangkan dengan tujuan penghematan waktu dan beaya untuk analisa secara rutin. Tehnik ini sangat menjanjikan tetapi memiliki keterbatasan karena adanya senyawa penghambat PCR yang secara alami terkandung di dalam sampel darah . Tujuan dari penelitian ini adalah membandingkan efektifitas antara uji PCR secara langsung dengan ekstraksi dan tanpa ekstraksi DNA untuk deteksi Brucella abortus di dalam darah. Tiga ( 3 ) sampel darah-EDTA yang berasal dari sapi penderita brucellosis dan 5 sampel darah-EDTA dari sapi potong yang mengalami abortus digunakan sebagai sampel dalam penelitian ini. Pasangan primer bcsp31 dan primer IS711 untuk amplifikasi gen dan species specific digunakan dalam penelitian. Hasil menunjukkan bahwa amplikon/pita pada posisi 223 bp dan 498 bp yang spesifik untuk Brucella abortus terdeteksi dari semua sampel yang dianalisa dengan gel agarosa 1,5%. Berdasarkan hasil penelitian dapat disimpulkan bahwa uji PCR secara langsung tanpa didahului dengan ekstraksi DNA merupakan tehnik yang sensitif, spesifik, sederhana, cepat dan murah untuk deteksi B. abortus di dalam sampel darah baik sapi perah maupun sapi potong dan oleh karena itu diharapkan dapat digunakan untuk memperbaiki program kontrol dan survailance yang telah ada untuk brucellosis. Kata kunci : brucellosis; PCR langsung; penghambat PCR; sampel darah-utuh; tanpa ekstraksi DNA

675. Evaluation of PLST for Clostridioides difficile Sequence-based Strain Typing

Background Clostridioides difficile is a leading cause of healthcare-associated infection (HAI), most often following antibiotic therapy. The source of these infections may be endogenous or nosocomial; effective intervention requires distinquishing between these, which in turn requires strain typing. Numerous methods have been developed for C. difficile typing, ranging from length-based ribotyping and MLVA to whole genome sequencing. However, none are routinely used in clinical settings due to low resolution, high cost, technical complexity, or requirement for cultured isolates. The application of polymorphic locus sequence typing (PLST) to epidemiological analysis of HAI and foodborne infections has recently been described; here this approach is extended to C. difficile. Methods Tandem repeats were bioinformatically identified in the genome sequence of ribotype 027 strain R20291. These were screened by BLASTN of GenBank databases for the most polymorphic locus, which identified CdMT1 (Mbp 3.149). DNA was purified from colonies or environmental (Banana Broth) cultures; bead-beating and PCR inhibitor removal steps were required for consistent results. Results CdMT1 encompassed MLVA repeat C6cd which, based on length alone, yielded the highest diversity index (DI) of 0.96. In contrast, CdMT1 sequence analysis yielded DI of >0.99. Comparison to ribotype further illustrated high level resolution; e.g., 9 ribotype 027 strains were resolved into 8 CdMT1 alleles. For initial laboratory evalulation, veterinary C. difficile isolates (44 canine, 4 bovine) were CdMT1 typed. Bioinformatic analysis of the 48 sequences resolved 24 CdMT1 alleles, including 8 clusters of 2 to 6 canine strains. Six of these clusters represented isolates from individual puppies in the same litter, or from different litters but the same household, while the bovine isolates formed a phylogenetically distinct group. Using the same DNA purification protocol, CdMT1 typing demonstrated compatibility with C. difficile-spiked stool samples and Banana Broth environmental cultures. Conclusion CdMT1 typing represents a potentially useful tool for outbreak detection and investigation in healthcare facilities, particularly in light of its compatibility with both stool and environmental samples. Disclosures Tom Edlind, PhD, MicrobiType LLC (Employee, Scientific Research Study Investigator)

Assessment of Commercial DNA Cleanup Kits for Elimination of Real-Time PCR Inhibitors in the Detection of Cyclospora cayetanensis in Cilantro

ABSTRACT Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration–validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method. HIGHLIGHTS

Optimizing an eDNA protocol for monitoring endangered Chinook Salmon in the San Francisco Estuary: balancing sensitivity, cost and time

Environmental DNA (eDNA) analysis has gained traction as a precise and cost effective method for species and waterways management. To date, publications on eDNA protocol optimization have focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of specific components of the eDNA protocol, such as water filtration and DNA extraction method when designing or choosing an eDNA pipeline. At the same time, these two parameters are essential for the experimental design of a project. Here we evaluate and rank different eDNA protocols in the context of Chinook salmon (Oncorhynchus tshawytscha) eDNA detection in an aquatic environment, the San Francisco Estuary. We present a comprehensive evaluation of multiple eDNA protocol parameters, balancing time, cost and DNA yield. For estuarine waters, which are challenging for eDNA studies due to high turbidity, variable salinity, and the presence of PCR inhibitors, we find that a protocol combining glass filters and magnetic beads, along with an extra step for PCR inhibitor removal, is the method that best balances time, cost, and yield. In addition, we provide a generalized decision tree for determining the optimal eDNA protocol for other studies on aquatic systems. Our findings should be applicable to most aquatic environments and provide a clear guide for determining which eDNA pipeline should be used for a given environmental condition.Author SummaryThe use of environmental DNA (eDNA) analysis for monitoring wildlife has steadily grown in recent years. Though, due to differences in the ecology of the environment studied and the novelty of the technique, eDNA currently shows a lack of standards compared to other fields. Here we take a deep look into each step of an eDNA assay, looking at common protocols and comparing their efficiencies in terms of time to process the samples, cost and how much DNA is recovered. We then analyze the data to provide a concise interpretation of best practices given different project constraints. For the conditions of the San Francisco Estuary we suggest the use of glass fiber filtration, the use of paramagnetic beads for DNA extraction and the use of a secondary inhibitor removal. We expect our findings to provide better support for managers to decide their standards ahead of project submission not only for estuarine conditions but for other waterine conditions alike.

Inhibition of Polymerase Chain Reaction: Pathogen-Specific Controls are better than Human Gene Amplification

PCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (ΔCp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0 %), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youden’s index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.